Novel glycero kinase, gene thereof and process for producing the glycerol kinase by using the gene

ABSTRACT

Relating to a gene encoding a new glycerol kinase and a method for preparing the enzyme by gene recombination technique. A Glycerol kinase which has high resistance against preservative, a recombinant vector comprising a gene encoding the glycerol kinase, a transformant prepared by transforming a host cell with the recombinant vector, and a method for producing the glycerol kinase, including culturing the transformant to produce glycerol kinase, and collecting the resulting glycerol kinase.

TECHNICAL FIELD

The present invention relates to a gene encoding a novel glycerol kinaseand a method for preparing the enzyme by a gene recombination technique.

BACKGROUND OF THE INVENTION

A glycerol kinase (EC 2. 7. 1. 30) is an enzyme catalyzing the reactionfor modifying glycerol into glycerol-3-phosphoric acid viaphosphorylation reaction depending on magnesium and ATP. Since aglycerol kinase had discovered in liver by Kalckar in 1937 (see forexample non-patent reference 1), it has been reported the purificationof glycerol kinase from such as rat liver, pigeon liver, Candidamycoderma, Cellulomonas flavigena, Thermus flavus (see for examplenon-patent references 2 to 5 and patent reference 1). It has been knownthat the glycerol kinase exists widely in general biological organisms.Additionally, it has also been reported that gene cloning from such ashuman, Bacillus subtilis, Saccharomyces cerevisiae and Thermus flavus(see for example non-patent references 6 to 9). The enzyme has beenstudied in detail in Escherichia coli in particular. In 1967, Hayashi etal. purified the enzyme (see for example non-patent reference 10). In1988, the cloning thereof was reported (see for example non-patentreference 11). Further, the enzyme has been studied in a wide rangeincluding research works on gene regulation and research works about theinhibition with allosteric inhibitors.

On the other hand, with regard to the industrial application of theglycerol kinase, the glycerol kinase is now used as a raw materialenzyme for clinical laboratory agents. In other words, neutral fat(triglyceride) in a sample is hydrolyzed with lipase to prepareglycerol, which is then modified into glycerol-3-phosphoric acid withthe enzyme. The resulting glycerol-3-phosphoric acid is used forassaying blood neutral lipid by calorimetric analysis using an oxidaseof glycerol-3-phosphoric acid and ultraviolet absorptiometry usingdehydrogenase of glycerol-3-phosphoric acid.

Recent clinical laboratory agents for biochemical tests have mainly beenlaboratory agents at solution state. Therefore, it is demanded that suchlaboratory agents in liquid should have high stability in addition tothe characteristic features (high reactivity with substrates, strictsubstrate specificity, etc.) traditionally demanded for enzymes. Variouscharacteristic features contributing to the stability of test agents inliquid can be suggested. Generally, preservative is added so as toenable long-term storage of test agents in liquid. Since suchpreservative may sometimes make enzymes unstable, high resistanceagainst preservative is one of desirable enzyme properties for testagents.

It has been believed so far that enzymes which have high thermalstability show high stability in test agents in liquid. Therefore, aglycerol kinase derived from thermophilic bacteria such as Bacillusstearothermophilus and Thermus flavus has been commonly used. However,such glycerol kinase has a problem of low resistance againstpreservative.

Patent reference 1: JP-A-56-121484

Non-patent reference 1: H. Kalckar, eds., “Enzymologia”, Vol.2, p. 47,1937

Non-patent reference 2: C. Bublitz, et al., “J. Biol. Chem.”, Vol. 211,p. 951, 1954

Non-patent reference 3: E. P. Kennedy, “Methods Enzymol.”, Vol. 5, p.476, 1962

Non-patent reference 4: H. U. Bergmeyer, et al., “Biochem.”, Vol. 333,p. 471, 1961

Non-patent reference 5: H. S. Huang, et al., “J. Ferment. Bioeng.”, No.83, p. 328, 1997

Non-patent reference 6: C. A. Sargent, et al., “Hum. Mol. Genet.”, Vol.3, p. 1317, 1994

Non-patent reference 7: C. Holmberg, et al., “J. Gen. Microbiol.”, Vol.136, p. 2367, 1990

Non-patent reference 8: P. Pavlik, et al., “Curr. Genet.”, Vol. 24, p.21, 1993

Non-patent reference 9: H. S. Huang, et al., “Biochim. Biophys. Acta”,Vol. 1382, p. 186, 1998

Non-patent reference 10: S. Hayashi, et al., “J. Biol. Chem.”, Vol. 242,p. 1030, 1967

Non-patent reference 11: D. W. Pettigrew, et al., “J. Biol. Chem.”, Vol.263, p. 135, 1988

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relation between the reaction pH of glycerol kinaseobtained in an Example of the present invention and the relativeactivity thereof (namely, optimum pH). Glycerol kinase activity wasassayed after reaction in each 50 mM buffer at 37° C. for 5 minutes. Thehorizontal axis shows pH, while the vertical axis shows relativeactivity. Black circles express the reaction in the presence of 50 mMMES buffer; black squares express the reaction in the presence of 50 mMHEPES buffer; black diamonds express the reaction in the presence ofTAPS buffer; black triangles express the reaction in the presence of 50mM CHES buffer; and white circles express the reaction in the presenceof 50 mM glycine-NaOH.

FIG. 2 shows the relation between the reaction temperature of glycerolkinase obtained in Example of the present invention and the relativeactivity thereof (namely, optimum temperature). Glycerol kinase activitywas assayed after reaction in 50 mM HEPES buffer, pH 7.9 at eachtemperature for 5 minutes. The horizontal axis shows temperature, whilethe vertical axis shows relative activity.

FIG. 3 shows the pH stability of glycerol kinase obtained in Example ofthe present invention. Glycerol kinase was dissolved in each of 50 mMbuffers to become about 10 U/ml and stored at 25° C. for 20 hours.Subsequently, glycerol kinase activity was assayed to determineremaining activity. The horizontal axis shows pH, while the verticalaxis shows remaining activity. Black circles expresse the reaction inthe presence of acetate buffer; black squares express the reaction inthe presence of potassium phosphate buffer; black diamonds express thereaction in the presence of CHES buffer; black triangles express thereaction in the presence of CAPS buffer.

FIG. 4 shows the thermal stability of glycerol kinase obtained in anExample of the present invention. Glycerol kinase was dissolved in 50 mMpotassium phosphate buffer of pH 7.5 to become about 10 U/ml and storedat each temperature for 15 minutes. Subsequently, glycerol kinaseactivity was assayed to determine the remaining activity. The horizontalaxis shows temperature, while the vertical axis shows remainingactivity.

FIG. 5 shows comparative data of remaining activity ratio (namely,resistance against preservative under storage at 4° C.) of the glycerolkinase obtained in an Example of the present invention and a glycerolkinase derived from various other microorganisms when they coexist withvarious preservatives at 4° C. After dissolving glycerol kinase fromeach origin in 50 mM potassium phosphate buffer of pH 7.5 to becomeabout 5 U/ml, each preservative was added at a concentration shown inthe figure and stored at 4° C. for one week, to assay the remainingactivity. The horizontal axis shows remaining activity ratio, while thevertical axis shows the origin of glycerol kinase. The remainingactivity ratio of the enzyme from each origin is shown in the order fromthe top as the activity when 0.3 mM Procline 300 is present, 0.8 nMProcline 150 is present, 500 mg/L IZU is present, 100 mg/L MIT ispresent and no preservative is added.

FIG. 6 shows comparative data of remaining activity ratio (namely,resistance against preservative under storage at 25° C.) of the glycerolkinase obtained in an Example of the present invention and a glycerolkinase derived from various other microorganisms when they coexist withvarious preservatives at 25° C. After dissolving glycerol kinase fromeach origin in 50 mM potassium phosphate buffer of pH 7.5 to becomeabout 5 U/ml, each preservative was added at a concentration shown inthe figure and stored at 25° C. for one week to assay the remainingactivity. The horizontal axis shows remaining activity ratio, while thevertical axis shows the origin of glycerol kinase. The remainingactivity ratio of the enzyme from each origin is shown in the order fromthe top as the activity when 0.3 mM Procline 300 is present, 0.8 mmProcline 150 is present, 500 mg/L IZU is present, 100 mg/L MIT ispresent and no preservative is added.

FIG. 7 shows comparative data of the thermal stability of the glycerolkinase obtained in an Example of the present invention and a glycerolkinase derived from Thermus flavus. After dissolving the glycerol kinasein 50 mM potassium phosphate buffer of pH 7.5 to become about 10 U/ml,glycerol kinases were stored at each temperature for 15 minutes to assaythe remaining activity. The horizontal axis shows temperature, while thevertical axis shows remaining activity ratio. Black circle expresseglycerol kinase of the present invention; black squares expressesglycerol kinase from Thermus fiavus.

FIG. 8 shows the result of the purification of glycerol kinase fromCellulomonas sp. JCM2471 in Reference Example.

FIG. 9 shows the result of purification of glycerol kinase obtained inExample of the present invention.

DISCLOSURE OF THE INVENTION

By isolating a gene encoding a novel glycerol kinase which has highresistance against preservative and establishing a method for producingthe enzyme by gene recombination technique, the enzyme can be applied tothe quantitative assay of neutral lipid and glycerol.

In order to solve the problems, the inventors of the present inventionmade investigations. Consequently, the novel glycerol kinase which hashigh resistance against preservative could successfully be isolated.Specifically, Cellulomonas sp. JCM2471 was isolated as a bacterium whichproduces such glycerol kinase. The glycerol kinase gene was successfullyisolated from the chromosomal DNA extracted from the bacterium, todetermine the whole nucleotide sequence of the DNA. Furthermore,glycerol kinase was highly produced in a transformant by generecombination technique to enable large-scale supply of glycerol kinaseof high purity at low cost. The bacterial strain can be purchased fromRiken Bioresource Center in Bioscience Technology Center, Riken.

In other words, the present invention provides glycerol kinase and thelike as described below.

Item 1 A glycerol kinase which has high resistance against preservative.

Item 2 The glycerol kinase according to item 1, wherein the resistanceagainst preservative expressed as a remaining activity ratio is 70% ormore when the glycerol kinase coexists with the 100 mg/L concentrationof preservative at 25° C. for one week.

Item 3 The glycerol kinase according to item 1 or 2, in which thepreservative is N-methylisothiazolone and/or a derivative thereof.

Item 4 The glycerol kinase according to item 1, which is a protein of(a) or (b) below:

-   -   (a) a protein consisting of an amino acid sequence represented        by SEQ ID NO:1 in the Sequence Listing; or    -   (b) a protein comprising an amino acid sequence of the amino        acid sequence (a) in which one or several amino acids are        deleted, substituted or added and having glycerol kinase        activity.

Item 5 A gene encoding a glycerol kinase which is a protein consistingof an amino acid represented by SEQ ID NO:1 in the Sequence Listing.

Item 6 A gene encoding glycerol kinase consisting of DNA of (c) or (d)below:

-   -   (c) a DNA consisting of a nucleotide sequence represented by SEQ        ID NO:2 in the Sequence Listing; or    -   (d) a DNA comprising a nucleotide sequence of the nucleotide        sequence (c) wherein one or several nucleotides are added,        deleted or substituted and encoding a protein having glycerol        kinase activity.

Item 7 A recombinant vector comprising a gene encoding the glycerolkinase according to any one of items 1, 2 or 3.

Item 8 A transformant comprising a host cell transformed with therecombinant vector according to item 7.

Item 9 A method for preparing a glycerol kinase, which comprisesculturing the transformant according to item 8 to produce a glycerolkinase, and collecting the glycerol kinase.

The glycerol kinase of the present invention comprises a glycerol kinasewhich has high resistance against preservative.

Additionally, the present invention relates to glycerol kinase whereinthe resistance against preservative, expressed as a remaining activityratio is 70% or more, preferably 80% or more, more preferably 90% ormore, when the glycerol kinase coexists preservative in concentration of100 mg/L at 25° C. for one week.

The resistance against preservative in the present invention can beevaluated on the basis of the remaining activity ratio when about 5 U/mLglycerol kinase coexists with preservative at 25° C. for one week in 50mM potassium phosphate buffer of pH 7.5.

The term preservative means a substance which is added to a reagentduring storage for the purpose of suppressing the growth ofmicroorganisms therein. The concentration of the preservative added isnot specifically limited, and preferably concentration which givessufficient effect. Reasonably, the concentration of a preservative to beadded varies depending on the type of the preservative and thecomposition of a reagent to be added. A person skilled in the art canappropriately determine suitable concentration of the preservative to beadded.

Taking account of concerns against the generation of resistant bacteriaduring the use of antibiotics, the use of antibiotics is consideredundesirable except for a case really demanding the use thereof.Additionally, no preservative effect may sometimes be obtained becauseof the influence of existing resistant bacteria. On the other hand, apreservative which can act on protein directly is more preferable, sinceit is difficult for microorganisms to acquire resistance against suchpreservative. Therefore, there is high possibility that suchpreservative may be used commonly in future. Such preservatives includesuch as N-methylisothiazolone (abbreviated as MIT) and/or a derivativethereof. Since such preservative which has direct actions on proteinalso work spontaneously coexisting enzyme protein, there is possibilitythat the preservative may make the enzyme protein unstable, depending onthe structure thereof. As described above, the resistance againstpreservative may be derived from both the function mechanism of thepreservative and the protein structure.

The gene encoding the glycerol kinase of the present invention may beextracted from microorganisms which produce glycerol kinase, for exampleCellulomonas sp. JCM2471 or may chemically be synthesized.

The gene includes such as (a) a DNA encoding the protein consisting ofthe amino acid sequence represented by SEQ ID NO:1, or (b) a DNAencoding the protein comprising an amino acid sequence in which one orseveral amino acids in the amino acid sequence (a) is deleted,substituted or added and having glycerol kinase activity. As to thedegree of the deletion, substitution or addition in the DNA, theresulting DNAs should have the fundamental characteristic properties ofthe intact DNA without any modification or should have improvement inthe characteristic properties of the intact DNA. The method forpreparing such variants is according to traditionally known methods.

Otherwise, the gene includes for example (c) a DNA consisting of thenucleotide sequence of SEQ ID NO:2, or (d) a DNA comprising a nucleotidesequence in which one or several nucleotides in the nucleotide sequence(c) is deleted, substituted or added and encoding a protein which hasglycerol kinase activity.

The method for obtaining the gene encoding the glycerol kinase of thepresent invention is described below as example. After separating andpurifying the chromosomal DNA of Cellulomonas sp. JCM2471, the DNA isfragmented using ultrasonic disruption or restriction treatment or thelike and ligated with a linear expression vector at the cohesive ends oradhesive ends of the both DNA fragments, by closing ring to construct arecombinant vector. The resulting recombinant vector thus obtained istransfected into a replicable host microorganism. Subsequently, theresulting transformants are screened using the expression of glycerolkinase activity to obtain a microorganism carrying the recombinantvector. Then, the microorganism is cultured to separate and purify therecombinant vector from the cultured microorganism and isolate theglycerol kinase gene from the recombinant vector.

The DNA from Cellulomonas sp. JCM2471 as a donor of the gene isspecifically collected as described below. Namely, the donormicroorganism is for example cultured under shaking for one day to threeday(s) and collected by the centrifugation. The resulting microorganismis subjected to lysis, to prepare a lytic material containing theglycerol kinase gene. As the lytic method, the microorganism is treatedwith for example lytic enzymes such as lysozyme and β-glucanase, incombination with protease, other enzymes and detergents such as sodiumlauryl sulfate (SDS), if necessary, and also in combination withphysical disruption processes such as freeze thawing and French pressprocess.

A separation and purification of the DNA from the lytic material thusobtained can be carried out by for example, appropriate combinations ofconventional processes including deproteinization such as phenoltreatment and protease treatment, ribonuclease treatment, and alcoholprecipitation treatment.

Further, the DNA can efficiently be obtained at high purify, usingvarious DNA extraction kits which is currently commercially available.

As the method for cleaving the DNA separated and purified from themicroorganism, for example, ultrasonification and restriction enzymetreatment can be carried out. Type II restriction enzymes which act onspecific nucleotide sequences are preferable.

As the vector, vectors constructed from phage or plasmid autonomouslyreplicable in host microorganisms for gene recombination are suitable.As the phage, for example, lambda ZAPII (manufactured by Stratagene),λgt·10, and λgt·11 can be used when Escherichia coli is a hostmicroorganism. As the plasmid, for example, pBR322, pUC19, pBluescript,pUCBM20, pUCBM21, pSE280 and pSE380 can be used when Escherichia coli(E. coli) is a host microorganism.

Such vector can be obtained by cleavage with restriction enzymes usedfor the cleavage of the microbial DNA as a donor of the glycerol kinasegene to prepare a vector fragment. However, the same restriction enzymeof the restriction enzyme used for cleaving the microbial DNA is notnecessarily used. As the method for conjugating the microbial DNAfragment to the vector DNA fragment, a known method using DNA ligase canbe carried out. For example, after annealing with the adhesive end ofthe microbial DNA fragment, a recombinant vector of the microbial DNAfragment and the vector DNA fragment is prepared by the use of anappropriate DNA ligase. After annealing, if necessary, the vector DNAfragment is transfected in a host microorganism to prepare a recombinantvector using a biological DNA ligase.

As the host microorganism, any microorganism can be used, as long as theresulting recombinant vector is stable and autonomously replicable toexpress an exogenous gene. Generally, for example, Escherichia coli (E.coli) strain K-12, Escherichia coli (E. coli) strain W3110, Escherichiacoli (E. coli) strain C600, Escherichia coli (E. coli) strain HB101, andEscherichia coli (E. coli) strain JM109 can be used. Additionally, avariant in which glycerol kinase is deficient is more preferably used asa host, although the activity of glycerol kinase derived from the hostis as low as negligible in a glycerol-free culture medium. Escherichiacoli (E. coli) strain KM1 may also be used.

As the method for transfecting the recombinant vector in a hostmicroorganism, competent cell method with calcium treatment andelectroporation can be used when Escherichia coli (E. coli) is a hostmicroorganism. Additionally, commercially available various Escherichiacoli-competent cells can also be used. By culturing the microorganismthus obtained as a transformant in a nutritious culture medium a greatamount of glycerol kinase is produced stably. As to the selection basedon the presence or absence of the transfection of the intendedrecombinant vector into a host microorganism, a microorganismsimultaneously expressing drug resistant markers of the vector carryingthe intended DNA and glycerol kinase activity can be screened for. Forexample, a microorganism growing on a selective culture medium based onthe drug resistant markers and producing glycerol kinase may beselected.

The nucleotide sequence of the glycerol kinase gene thus obtained by themethod can be analyzed by commercially available reagents and automaticsequencers based on the dideoxy method described in Science (Science,214, 1205-1210, 1981) and improved methods thereof. Additionally, theamino acid sequence of glycerol kinase was assumed on the basis of thedetermined nucleotide sequence. The recombinant vector carrying theglycerol kinase gene as once selected can be recovered from thetransformant microorganism, which can easily be transfected into anothermicroorganism. Additionally, DNA as the glycerol kinase gene can berecovered from the recombinant vector carrying the glycerol kinase geneby restriction enzyme and PCR method, which is then conjugated toanother vector fragment and transfected into a host microorganismeasily.

As the mode for culturing a host microorganism as the transformant, theculture conditions therefore can be selected, taking account of thenutritional and physiological properties of the host. Generally, in mostcases, the host microorganism is cultured in liquid. Industrially, suchhost microorganism is advantageously aeration cultured with aeration andshaking. As a carbon source in the culture medium, carbon sourcesusually used for microbial culture are widely used. Any carbon sourcesassimilable by host microorganisms can be used and include such asglucose, sucrose, lactose, maltose, fructose, molasses, and pyruvicacid. As a nitrogen source, nitrogen compounds which can be used by thehost microorganism can be used and include such as organic nitrogencompounds such as peptone, meat extract, casein hydrolysate, and soybeanbran alkali extracts; and inorganic nitrogen compounds such as ammoniumsulfate and ammonium chloride. Besides, salts such as phosphate salts,carbonate salts, sulfate salts, salts of magnesium, calcium, potassium,iron, manganese and zinc, specific amino acids, and specific vitaminscan be used if necessary.

The culture temperature can appropriately be changed within a rangewherein the host microorganism grows and glycerol kinase is produced. Incase of Escherichia coli (E. coli), preferably, the culture temperatureis preferably about 20 to 42° C. The culturing time changes more orless, depending on the culture conditions. Culturing may be terminatedin appropriate timing when the yield of glycerol kinase is estimated toreach maximum. Generally, the time is about 20 to 48 hours. The pH ofthe culture medium can appropriately be changed within a range whereinthe host microorganism to grow and for glycerol kinase is produced.Generally, preferably, the pH is about 6.0 to 9.0.

The method for recovering the bacterial cell from the liquid culture isin accordance with methods generally used. For example, the bacterialcell can be recovered by centrifugation or filtration. In case thatglycerol kinase in the liquid culture is extracellularly secreted, asolution separated from the bacterial cell may can be used. According tothe following method after the disruption of the bacterial cell,glycerol kinase can be separated and purified. In case that glycerolkinase exists intracellularly, glycerol kinase can be extracted viaenzymatic or physical disruption methods as described above. A fractionof glycerol kinase is recovered from the crude enzyme extract solutionthus obtained by for example ammonium sulfate precipitation. The crudeenzyme solution is generally desalted by routine purification methods,for example dialysis using semi-permeable membrane or gel filtration onSephadex G-25 (Amersham Biosciences).

After the procedure, a crude enzyme specimen can be obtained byseparation and purification with phenyl Sepharose First Flow (AmershamBiosciences) column chromatography and DEAE-Sepharose First Flow(Amersham Biosciences) column chromatography. The resulting purifiedenzyme specimen is purified at such a degree that the specimen shows analmost single band by electrophoresis (SDS-PAGE).

The protein which has glycerol kinase activity obtained by the method ofthe present invention has the following physico-chemical propertiesdescribed below.

-   (1) Function: Glycerol+ATP    Glycerol-3-phophoric acid+ADP-   (2) Optimal pH: about 10.0-   (3) Optimal temperature: about 50° C. (reaction in 20 mM HEPES    buffer, pH 7.9 for 5 minutes)-   (4) pH stability: about 6.0-10.0 (the range involving the remaining    activity of 90% or more even after 20 hr treatment at 25° C.)-   (5) Thermal stability: about 45° C. or less (the range involving the    remaining activity of 90% or more in 50 mM potassium phosphate    buffer, pH 7.5 even after 15-min treatment)-   (6) Molecular weight: about 55,000 (SDS-PAGE), about 176,000 (gel    filtration)-   (7) Km value: about 6.9×10⁻⁶ M (glycerol), about 1.11×10⁻⁴ M (ATP)-   (8) Relative activity: about 41.2/mg-   (9) The remaining activity ratios under storage at 4° C. for one    week and at 25° C. for one week when the protein coexists with 100    mg/L MIT in 50 mM potassium phosphate buffer of pH 7.5 was almost    100% (FIG. 5) and about 92% (FIG. 6), respectively.

The glycerol kinase of the present invention may exist in any form, withno specific limitation. If necessary, the glycerol kinase of the presentinvention may be in a freeze-dried form, a liquid form or any otherforms. In case of freeze-drying, additionally, suitable excipients,stabilizers and the like may be added. In case of the liquid form,furthermore, suitable buffers and/or other ingredients may be added.

BEST MODE FOR CARRYING OUT THE INVENTION

In the following Examples, the activity of glycerol kinase was assayedas follows. ATP was purchased from Oriental Yeast Co., Ltd. Bovine serumalbumin was purchased from Sigma Aldrich. Oxidase ofglycerol-3-phosphoric acid (Code No. G30-301) and peroxidase (Code No.PEO-301) manufactured by Toyobo Co., Ltd. were used. Other reagents werepurchased from Nakarai Tesque for use.

<Assay Method 1: Method for Assaying Glycerol Kinase Activity by RateAssay>

Generally, the activity was assayed by the method. Using glycerol assubstrate, the enzyme activity was assayed on the basis of the amount ofproduced glycerol-3-phosphoric acid. By adding 0.1 M HEPES buffer of pH7.9 to 0.2 ml of 0.5% 4-aminoantipyrine solution, 0.2 ml of 1.5% phenolsolution, 200 U of the oxidase of glycerol-3-phosphoric acid, 80 U ofperoxidase, and 48.4 mg of ATP, a total volume is adjusted to be 21 ml.The resulting solution was used as the stock solution for the followingassays. To 3 ml of the assay stock solution, 50 μl of 0.3 M glycerolsolution and 100 μl of the enzyme solution were added and mixed. Eachreaction was assayed by recording the absorbance at 500 nm for 3 minuteswith a spectrophotometer which is controlled to be 37° C., andcalculating the change of the absorbance per minute on the initiallinear part (triangle OD test). A blank test was conducted by adding 100μl of a diluted enzyme solution (20 mM potassium phosphate buffer of pH7.5 containing 0.2% bovine serum albumin) instead of the enzyme solutionand carrying out the same procedures as described above and calculatingthe change of the absorbance per minute (triangle OD blank).

Based on the change of the absorbance as obtained, the enzyme activityof glycerol kinase was calculated according to the following formula.Additionally, one unit (1 U) of the enzyme was defined as an amountthereof required for the phosphorylation of 1 μmol glycerol per minuteunder the aforementioned conditions.Activity value (U/ml)=[triangle OD/min (triangle ODtest-triangleODblank)×3.15 (ml)×dilution ratio]/[13.3×½×1.0 (cm)×0.1 (ml)]  Formula

3.15 ml=volume of reaction mixture solution

13.3=extinction coefficient per millimole quinone dye under theaforementioned assay conditions

½=coefficient due to the quinone dye amount formed from one molecule ofaqueous hydrogen peroxide produced via the enzyme reaction, which is ½molecule.

1.0 cm=optical path of cell

0.1 ml=volume of enzyme sample

<Assay Method 2: Method for Assaying Optimal Temperature>

The method was used so as to find the optimal temperature for theenzyme.

After dividing 3 ml of an active reaction solution (20 mM HEPES bufferof pH 7.9 containing 4 mM ATP and 2 mM magnesium chloride) in a testtube, 0.1 ml of a glycerol kinase solution after dilution to anappropriate concentration (about 1 U/ml of the activity measuredaccording to the assay method 1) was added thereto and mixed thoroughly.Then, the reaction mixture is preliminarily heated at each temperaturefor about 3 minutes. Then, 0.05 ml of aqueous 0.3 M glycerol solutionwas added and mixed for initiating the reaction. After the reaction forexact 10 minutes, 1 ml of 1N hydrochloric acid was added to terminatethe enzyme reaction. After adding 0.15 ml of the reaction-terminatedsolution to 3 ml of a chromogenic solution (0.2 M HEPES buffer of pH 7.9containing 0.01% 4-aminoantipyrin, 0.02% phenol, 5 U/ml peroxidase and16 U/ml glycerol-3-phosphoric acid oxidase) and mixing, reaction at 37°C. for about 5 minutes was carried out to measure the absorbance at 500nm. Then, the absorbance of 1 mM L-glycerol-3-phosphoric acid solutionwas simultaneously measured. The amount of L-glycerol-3-phosphoric acidproduced via each of the reactions was determined. Herein, a blank testwas carried out by determining the amount of L-glycerol-3-phosphoricacid produced in an unspecific manner at each reaction temperature,using the diluted enzyme solution instead of the glycerol kinasesolution.

<Assay Method 3: Method for Assaying Optimal pH>

The method was used so as to examine the optimal pH of the enzyme.

After dividing 3 ml of an active reaction solution (50 mM buffer at eachpH, containing 4 mM ATP and 2 mM magnesium chloride) in a test tube, 0.1ml of a glycerol kinase solution after dilution to an appropriateconcentration (about 1 U/ml of the activity measured according to theassay method 1) was added thereto and mixed throughly. Then, thereaction mixture is preliminarily heated at 37° C. for about 3 minutes.Then, 0.05 ml of 0.3 M glycerol solution was added and mixed forinitiating the reaction. After reaction for exact 10 minutes, 1 ml of 1Nhydrochloric acid was added to terminate the enzyme reaction. Afteradding 0.15 ml of the reaction-terminated solution to 3 ml of achromogenic solution (0.2 M HEPES buffer of pH 7.9 containing 0.01%4-aminoantipyrin, 0.02% phenol, 5 U/ml peroxidase and 16 U/mlglycerol-3-phosphoric acid oxidase) and mixing, reaction at 37° C. forabout 5 minutes was carried out to measure the absorbance at 500 nm.Then, the absorbance of 1 mM L-glycerol-3-phosphoric acid solution wassimultaneously measured. The amount of L-glycerol-3-phosphoric acidproduced via each enzyme reaction was determined. Herein, a blank testwas carried out by determining the amount of L-glycerol-3-phosphoricacid produced in the buffer at each pH in an unspecific manner withoutany enzymatic reaction, using the diluted enzyme solution instead of theglycerol kinase solution.

REFERENCE EXAMPLE

Purification of Glycerol Kinase from Cellulomonas sp. JCM2471

After inoculating one platinum loop of Cellulomonas sp. JCM2471 in 60 mlof the LB liquid culture medium (in 500-mL Sakaguchi's flask), culturingwith shaking at 30° C. overnight was carried out. The liquid culture waswholly inoculated in a 6 L of culture medium for glycerol kinaseproduction (10 L-jar fermenter, 2% glycerol, 2% polypeptone(manufactured by Nihon Pharmaceutical Co., Ltd.), 0.2% yeast extract(manufactured by Oriental Yeast Co., Ltd.), 0.2% NaCl, 0.02% magnesiumsulfate, 0.7% dipotassium phosphate, pH 7.3) and cultured with shakingand aeration at 35° C. for about 20 hours. The amount of glycerol kinaseproduced per the culture medium was about 3 U/ml.

The bacterial cell was recovered by centrifugation from the liquidculture, suspended in 50 mM potassium phosphate buffer of pH 7.5 anddisrupted with glass beads using a Dinomill disrupter to extractglycerol kinase, which was used as crude enzyme solution. Ammoniumsulfate was added to the crude enzyme solution, from which a 35-55%saturated fraction was recovered. This fraction after salting out wasdesalted by gel filtration with Sephadex G-25 (Amersham Biosciences),and subjected sequentially to DEAE Sepharose CL-6B (AmershamBiosciences) column chromatography, phenyl Sepharose CL-6B (AmershamBiosciences) column chromatography, gel filtration with Sephadex G-25and DEAE Sepharose CL-6B column chromatography, to recover a purifiedenzyme specimen. The purification results are shown in FIG. 8.

The protein purified by the aforementioned method shows an almostuniform band on SDS-PAGE, and had a relative activity of about 40.9 U/mgprotein. Additionally, the protein concentration was approximatelycalculated by defining that 1 mg/ml protein concentration correspondedto 1 Abs absorbance of the enzyme solution at 280 nm.

Additionally, the molecular weight of the subunit estimated by SDS-PAGEwas about 55,000. Further, the N-terminal amino acid sequence wasanalyzed by an amino acid sequencer based on the principle Edmandegradation method. The sequence from the N terminus wasAla-Asp-Tyr-Val-Leu-Ara-Ile.

Example 1

Separation of Chromosomal DNA from Cellulomonas sp. JCM2471

The chromosomal DNA of Cellulomonas sp. JCM2471 was separated by thefollowing method. One platinum loop of the bacterial strain wasinoculated in an LB culture broth (5 ml charged amount/30-ml test tube;1.0% polypeptone, 0.5% yeast extract, 1.0% NaCl, pH 7.4) and culturedwith shaking at 30° C. for overnight. The bacterial cell was recoveredfrom 1 ml of the bacterial cell by centrifugation (12,000 rpm, 10minutes, 4° C.). The chromosomal DNA was extracted from the recoveredbacterial cell, using MagExtractor-genome kit (manufactured by ToyoboCo., Ltd.) according to the procedures described in the manual. Thechromosomal DNA was obtained at about 20 μg from 1 ml of the bacterialcell.

Example 2

Amplification of Glycerol Kinase Gene by PCR

Primers for polymerase chain reaction (PCR) were prepared on the basisof the nucleotide sequences of glycerol kinase from Escherichia coli (E.coli), Bacillus subtilis, and Pseudomonas aeruginosa, of which theircloning is currently reported. The nucleotide sequences of SEQ ID NO:3in the sequence listing and SEQ ID NO:4 in the sequence listing show thePCR primers. By mixing 100 ng of the DNA obtained in Example 1, 200 pmolof each of the primers, 10 μl of a dNTP mixture, 10 μl of a reactionbuffer, and 2.5 U of AmpliTaq DNA polymerase (manufactured by PerkinElmer), a final volume of reaction liquid mixture was adjusted to be 100μl. This was subjected to PCR including repeating 30 times of a cycle ofa modification reaction at 94° C. for one minute, an annealing reactionat 45° C. for one minute and an extension reaction at 72° C. for 3minutes. Consequently, a fragment of about 800 bp as an intended sizewas amplified. The nucleotide sequence of the PCR product wasdetermined. When the speculated amino acid sequence was compared withthe amino acid sequence of glycerol kinase from Pseudomonas aeruginosa,high homology was shown. Thus, it was indicated that a part of theintended glycerol kinase gene was amplified.

Example 3

Cloning of Full-Length Glycerol Kinase Gene

About 2 μg of the chromosomal DNA obtained in Example 1 was digestedwith various restriction enzymes, separated by 0.7% agarose gelelectrophoresis and transferred onto a nitrocellulose filter. The filterwas subjected to Southern hybridization according to the protocolattached to the reagents, using the PCR product obtained in Example 2 asprobe and ECL direct nucleic acid labeling and detection system(Amersham Biosciences), to screen for fragments of the glycerol kinasegene. Consequently, the DNA fragment containing the full-length glycerolkinase gene was detected as a fragment of about 6.5 kb after cleavagewith KpnI (manufactured by Toyobo Co., Ltd.) and NotI (manufactured byToyobo Co., Ltd.).

Then, the DNA fragment was recovered from the agarose gel usingMagExtractor-PCR & gel clean up-kit (manufactured by Toyobo Co., Ltd.)according to the procedures described in the manual. Alternatively, 0.5μg of puCBM21 (Boehringer Mannheim) was similarly cleaved with KpnI andNotI, for dephosphorylation treatment with bacterial alkali phosphatase(manufactured by Toyobo Co., Ltd.). Subsequently, both the DNA fragmentsreacted together at 16° C. for one hour using Ligation High kit(manufactured by Toyobo Co., Ltd.) for ligation to transform thecompetent cell of Escherichia coli JM 109 (manufactured by Toyobo Co.,Ltd.). The transformant was obtained by spreading the resulting mixtureon an LB agar culture medium containing 100 μg/ml ampicillin andculturing overnight at 37° C. This recombinant vector was designated aspCGK1.

Example 4

Determination of Nucleotide Sequence of Gene of Enzyme Glycerol Kinase

Concerning the nucleotide sequence of the glycerol kinase gene cloned inpCGK1, the DNA sequence was determined starting from both the ends ofthe inserted gene using common primers for sequencing of pUC-seriesvectors and big diterminator cycle sequencing FS ready reaction kit(manufactured by Applied BioSystems) and ABI PRISM 310 Genetic Analyzer(manufactured by Perkin Elmer). Further, additional primers wereprepared on the basis of the determined sequence to determine thefull-length inserted DNA sequence by primer walking.

The DNA sequence corresponding to the open reading frame of thedetermined glycerol kinase gene and the amino acid sequence speculatedon the basis of the DNA sequence are shown as SEQ ID NO:2 in thesequence listing. Additionally, the sequence of seven residues in theamino acid sequence including Ala in the second residue speculated fromthe DNA sequence was completely corresponds to the result of the aminoacid sequencing of the purified enzyme. Furthermore, the molecularweight of glycerol kinase as calculated on the basis of the speculatedamino acid sequence from which methionine as the initiation codon waseliminated was 55, 142. The molecular weight corresponds well with themolecular weight of the enzyme purified from Cellulomonas sp. JCM2471which is determined by SDS-PAGE analysis.

Example 5

Construction of Glycerol-Deficient Host

Primers of SEQ ID NO:5 in the sequence listing and SEQ ID NO:6 in thesequence listing were prepared on the basis of the nucleotide sequenceof glycerol kinase derived from Escherichia coli (E. coli) which isregistered on the GenBank database. Furthermore, the chromosomal DNA ofEscherichia coli (E. coli) strain K12 was obtained by the same method asin Example 1.

By mixing 100 ng of the chromosomal DNA, 200 pmol of each of theprimers, 10 μl of 2mM dNTP mixture, 10 μl of a reaction buffer, and 2.5U of AmpliTaq DNA polymerase (manufactured by Perkin Elmer), a finalvolume of the mixture was adjusted to be 100 μl. This was subjected toPCR by carrying out 41 times of a cycle of a modification reaction at94° C. for 3 minutes, a modification reaction at 98° C. for 30 secondsand an annealing/extension reaction at 68° C. for 3 minutes and thencarrying out one cycle of a modification reaction at 98° C. for 30seconds, an annealing/extension reaction at 68° C. for 3 minutes and anextension reaction at 72° C. for 10 minutes. Consequently, a PCR productof about 1.5 kbp in the same size as that of the intended gene wasobtained.

After the PCR product was purified using MagExtractor-PCR & gel cleanup-kit according to the procedures described in the manual, the reactionof the PCR product of about 0.2 μg with 0.5 μg of pUC19 cleaved with arestriction enzyme SmaI was carried out at 16° C. for one hour usingLigation High kit to transform the competent cell of Escherichia coli(E. coli) JM 109. The transformant was obtained by spreading theresulting mixture on an LB agar culture medium containing 100 μg/mlampicillin and cultured overnight at 37° C. This recombinant vector wasdesignated as pUCGK.

The pUCGK was cleaved with a restriction enzyme BstEII (manufactured byToyobo Co., Ltd.). The cleavage ends thereof were made to be blunt endedusing Blunting High kit (manufactured by Toyobo Co., Ltd.), according tothe procedures described in the manual. On the other hand, pUCK4(manufactured by Amersham Biosciences) was cleaved with HincII. Afterseparating DNA fragments containing the kanamycin resistant gene byagarose gel electrophoresis, the DNA fragments was purified andrecovered by using MagExtractor-PCR & gel clean up-kit. Reaction of theboth fragments was carried out at 16° C. for one hour, using LigationHigh kit to transform the competent cell of E. coli JM 109. Thetransformant was obtained by spreading the resulting mixture on an LBagar culture medium containing 100 μg/ml ampicillin and 50 μg/mlkanamycin for overnight culturing at 37° C. The resulting recombinantvector was designated as pUCGKm.

Furthermore, pUCGKm was cleaved with restriction enzymes EcoRI(manufactured by Toyobo Co., Ltd.) and SalI (manufactured by Toyobo Co.,Ltd.) to separate a fragment containing the glycerol kinase gene and thekanamycin resistant gene by agarose gel electrophoresis. The fragmentwas purified and recovered by using MagExtractor-PCR & gel clean up-kit.On the other hand, a temperature sensitive plasmid pCH02 (a derivativefrom pSC101 plasmid; S. Matsuyama, et al., J. Mol. Biol., 175, 331(1984)) was also cleaved with EcoRI (manufactured by Toyobo Co., Ltd.)and SalI (manufactured by Toyobo Co., Ltd.), and then reacted with thefragment containing the glycerol kinase gene and the kanamycin resistantgene at 16° C. for one hour with Ligation High kit to transformEscherichia coli (E. coli) strain K-12 by electroporation using GenePulsar (manufactured by Bio-Rad). Herein, the conditions for theelectroporation is in accordance with the conditions for Escherichiacoli (E. coli) which is described in the manual for Gene Pulsar. Thetransformant was obtained by spreading the resulting mixture on an LBagar culture medium containing 100 μg/ml ampicillin and 50 μg/mlkanamycin for overnight culturing at 30° C.

The resulting transformant was inoculated in an LB culture brothcontaining 50 μg/ml kanamycin (5 ml charged volume/20 ml-test tube) andcultured with shaking at 37° C. for 24 hours. The liquid culture wassubcultured in a fresh LB culture broth containing kanamycin, repeatedlyfour times. The liquid culture was diluted with sterile physiologicalsaline, spreaded on an LB agar culture medium containing 50 μg/mlkanamycin to separate single colonies, from which a colony sensitive toampicillin and resistant against kanamycin was separated and defined asEscherichia coli (E. coli) strain KM1 (E. coli KM1).

The Escherichia coli strain KM1 (E. coli KM1) was inoculated in TrafficBroth containing 50 μg/ml kanamycin (5 ml charged volume/20-ml testtube; 1.2% polypeptone, 2.4% yeast extract, 0.5% glycerol, 0.231%monopotassium phosphate, 1.254% dipotassium phosphate), for shakingculture at 37° C. for 24 hours. Bacterial cells were recovered from 1 mlof the liquid culture by centrifugation and suspended in 1 ml of 50 mMpotassium phosphate buffer of pH 7.5. The bacterial cells in theresulting suspension were disrupted with an ultrasonic disrupter, andcentrifuged. Although the resulting supernatant was used as a crudeenzyme to assay the activity of glycerol kinase, no significant enzymeactivity was detected.

Example 6

Construction of Glycerol Kinase Expression Vector pCGK12

pCGK1 was cleaved with restriction enzymes NcoI (manufactured by ToyoboCo., Ltd.) and NotI, to separate a DNA fragment of about 2 kb by 1%agarose gel electrophoresis, which contained the glycerol kinase gene.Subsequently, the DNA fragment was recovered from the agarose gel usingMagExtractor-PCR & gel clean up-kit according to the proceduresdescribed in the manual. On the other hand, 0.5 μg of pSE380(manufactured by Invitrogen) was cleaved with NcoI and NotI, and treatedwith bacterial alkali phosphatase (manufactured by Toyobo Co., Ltd.) fordephosphorylation. Subsequently, both the DNA fragments reacted withLigation High kit at 16° C. for one hour for ligation, to transform thecompetent cell of Escherichia coli (E. coli) strain JM109. Thetransformant was obtained by spreading the resulting mixture on an LBagar culture medium containing 100 μg/ml ampicillin and culturedovernight at 37° C. This recombinant vector was designated as pCGK2.

Then, Escherichia coli strain JM109 (E. coli JM109) transformed withpCGK12 was inoculated in an LB culture broth (5 ml/30-ml charged intotest tube) containing 100 μg/ml ampicillin and cultured with shaking at37° C. overnight. After completion of the culturing, the liquid culturewas centrifuged to recover bacterial cells, from which pCGK12 waspurified using MagExtractor-Plasmid-kit (manufactured by Toyobo Co.,Ltd.). The purified plasmid of about 20 μg was recovered from 5 ml ofthe bacterial cells.

Furthermore, pCGK12 was adjusted to a concentration of 0.05 μg/>l andintroduced into Escherichia coli strain KM1 (E. coli KM1) which is aglycerol kinase-deficient strain as a host, by electroporation usingGene Pulsar. The transformant was selected in an LB agar culture mediumcontaining 100 μg/ml ampicillin and 50 μg/ml kanamycin, to screen for acolony which is simultaneously resistant against the two types ofantibiotics as a transformant after overnight culturing at 30° C. Theefficiency of transformation then was about 1×10⁶ cfu/μg-DNA.

Herein, Escherichia coli strain KM1 (pCGK12) (E. coli KM1 (pCGK12)) asthe transformant was deposited under an accession number of FERM P-18992at International Patent Organism Depository, National Institute ofAdvanced Industrial Science and Technology on Sep. 3, 2002.

Example 7

Expression of Recombinant Glycerol Kinase and Recovery of PurifiedEnzyme

One platinum loop of the transformant obtained in Example 6 wasinoculated in an LB culture medium (5 ml charged volume/20-ml test tube)containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultured withshaking at 30° C. overnight, to obtain a seed liquid culture. The liquidculture was inoculated at 1% in 1 L-Traffic Broth (250 ml chargedvolume)/2 L-Sakaguchi's flask per one bottle) containing 100 μg/mlampicillin and 50 μg/ml kanamycin, and cultured with shaking at 37° C.for 20 hours. At the time of the completion of the culturing, theglycerol kinase activity was about 6.8 U/ml of liquid culture.

After recovering the bacterial cells from the liquid culture bycentrifugation, the bacterial cells were suspended in 50 mM potassiumphosphate buffer of pH 7.5 and homogenized with French press.Subsequently, NaCl was dissolved in the solution of the homogenizedbacterial cells to become 0.1 M to which 5% polyethylene imine solutionwas added to 0.5% of the solution. Then, insoluble matters were removedfrom the resulting mixture by centrifugation. The resulting supernatantwas defined as crude enzyme solution.

Subsequently, ammonium sulfate was added to the crude enzyme solution to60-% saturation for salting out. The resulting precipitate was recoveredby centrifugation, and then dissolved again in 50 mM potassium phosphatebuffer, pH 7.5. Furthermore, the resulting solution was subjected todesalting process by gel filtration by Sephadex G-25 (AmershamBiosciences). Thereafter, the resulting solution was applied to HiTrap QHP column (Amersham Biosciences), rinsed with 0.2 M NaCl, and eluted ona linear 0.2 M-0.6 M NaCl gradient.

Further, fractions with the glycerol kinase activity were collected, towhich ammonium sulfate was added to 20-% saturation. Insoluble matterswere removed by centrifugation. The enzyme solution was applied toHiTrap Phenyl FF column (Amersham Biosciences) buffered with 50 mMpotassium phosphate buffer of pH 7.5 containing ammonium sulfate at 20%saturation, rinsed with the same buffer and eluted by a linear gradientof ammonium sulfate from 20% saturation to 0% saturation. The fractionof the glycerol kinase activity was recovered and desalted by SephadexG-25 gel filtration, to recover a purified enzyme sample. Thepurification results are shown in FIG. 9.

The protein purified by the method indicated an almost uniform band witha relative activity of about 41.2 U/mg-protein. The concentration of theprotein was calculated approximately in the same manner as in ReferenceExample 1. The molecular weight of the subunit estimated by SDS-PAGE wasabout 55,000, while the molecular weight of the intact enzyme after gelfiltration with TSK-G3000 SW (7.6-mm diameter and 30-cm heightmanufactured by Tosoh Corporation) was about 176,000.

The glycerol kinase obtained by the method had the followingcharacteristic features.

-   (1) Function: Catalyzing the following reaction.    Glycerol+ATP    Glycerol-3-phosphoric acid+ADP-   (2) Working pH: Relation between reaction pH and relative activity    is shown in FIG. 1.-   Optimal pH was about 10.0.-   (3) Working temperature: Relation between reaction temperature and    relative activity is shown in FIG. 2.-   Optimal temperature: about 50° C. (reaction in 20 mM HEPES buffer of    pH 7.9 for 5 minutes)-   (4) pH stability: pH stability is shown in FIG. 3.-   Stable at about 6.0 to 10.0 (within the range, 90% or more of the    activity remains even at 25° C. for 20 hours).-   (5) Thermal stability: Thermal stability is shown in FIG. 4.

Stable at about 45° C. or less (within the range, 90% or more of theactivity remains even after 15-min treatment in 50 mM potassiumphosphate buffer of pH 7.5).

-   (6) Molecular weight: about 55,000 (SDS-PAGE), about 176,000 (gel    filtration)-   (7) Km value: about 6.9×10⁻⁶ M (glycerol), about 1.11×10⁻⁴ M (ATP)

The Km values of glycerol and ATP were calculated according to theLineweaver-Burk equation according to the method described in thesection “Method for assaying glycerol kinase activity” <method 1>,wherein the concentration of glycerol or ATP was changed to assay theglycerol kinase activity at each substrate concentration.

-   (8) Relative activity: About 41.2 U/mg-   (9) Resistance against the presence of preservative: Remaining    activity ratio in coexistence with 100 mg/L MIT in 50 mM potassium    phosphate buffer of pH 7.5 is shown in FIGS. 5 and 6. The activity    was assayed according to the method 1.

The remaining activity ratios were almost 100% after storage at 4° C.for one week and about 92% after storage at 25° C. for one week. Theremaining activity ratio in coexistence with other preservatives arealso shown. Glycerol kinase from other origins as comparative subjectwas purchased from Sigma Aldrich Japan, except for glycerol kinase fromThermus flavus (manufactured by Toyobo Co., Ltd.). Additionally,N-methylisothiazolone (abbreviated as MIT) and imidazolidinylurea(abbreviated as IZU) among the preservatives used were purchased fromRoche Diagnostics, while ProClin 150 and ProClin 300 were purchased fromSigma Aldrich, Japan.

The glycerol kinase of the present invention kept a remaining activityratio of 90% or more even after storage at 25° C. for one week.

Additionally, FIG. 7 shows comparative thermal stability data with thatfrom Thermus flavus as comparative example. Although the glycerol kinasederived from Thermus flavus had apparently high thermal stabilitycompared with the glycerol kinase of the present invention, the glycerolkinase of the present invention is shown to have high stability in thecoexistence of preservatives. It is thus indicated that the glycerolkinase of the present invention has high resistance againstpreservatives.

INDUSTRIAL APPLICABILITY

In accordance with the present invention, the gene encoding new glycerolkinase which has high resistance against preservatives compared withknown glycerol kinase was isolated, to establish a method for preparingthe enzyme by gene recombination technique, which can be applied toglycerol assay.

1. A Glycerol kinase which has high resistance against preservative. 2.The glycerol kinase according to claim 1, wherein the resistance againstpreservative expressed as a remaining activity ratio is 70% or more whenthe glycerol kinase coexists with the preservative at 25° C. for oneweek.
 3. The glycerol kinase according to claim 1 or 2, in which thepreservative is N-methylisothiazolone and/or a derivative thereof. 4.The glycerol kinase according to claim 1, which is a protein of (a) or(b) below: (a) a protein consisting of an amino acid sequencerepresented by SEQ ID NO: 1 in the Sequence Listing; or (b) a proteincomprising an amino acid sequence of the amino acid sequence (a) inwhich one or several amino acids are deleted, substituted or added andhaving glycerol kinase activity.
 5. A gene encoding a glycerol kinasewhich is a protein consisting of an amino acid represented by SEQ ID NO:1 in the Sequence Listing.
 6. A gene encoding glycerol kinase consistingof DNA of (c) or (d) below: (c) a DNA consisting of a nucleotidesequence represented by SEQ ID NO:2 in the Sequence Listing; or (d) aDNA comprising a nucleotide sequence of the nucleotide sequence (c)wherein one or several nucleotides are added, deleted or substituted andencoding a protein having glycerol kinase activity.
 7. A recombinantvector comprising a gene encoding the glycerol kinase according to anyone of claims 1 or
 2. 8. A transformant comprising a host celltransformed with the recombinant vector according to claim
 7. 9. Amethod for preparing a glycerol kinase, which comprises culturing thetransformant according to claim 8 to produce a glycerol kinase, andcollecting the glycerol kinase.
 10. A recombinant vector comprising agene encoding the glycerol kinase according to claim
 3. 11. Atransformant comprising a host cell transformed with the recombinantvector according to claim
 10. 12. A method for preparing a glycerolkinase, which comprises culturing the transformant according to claim 11to produce a glycerol kinase, and collecting the glycerol kinase.